Project Summary. The goal of this project is to define the interactions between RNA polymerase II, the basal transcription factors, and the chromatin template that lead to accurate transcription initiation and productive elongation. Using a combination of approaches in the yeast Saccharomyces cerevisiae model system, several fundamental aspects of gene expression will be studied. In the first specific aim, initiation and elongation complexes formed under various conditions will be purified on immobilized chromatin templates and then analyzed by quantitative mass spectrometry. It is possible some new factors associated with transcription complexes will be identified, but what is of greater interest is the exchange of factors that occur at various stages of transcription. Using various mutant or depleted extracts, factor interdependencies will be revealed. We will ask whether different activators recruit distinct co-activators, and how different promoters and transcribed sequences influence the associated proteins. The second specific aim will probe the function of the Set1/COMPASS complex, which methylates histone H3 at lysine 4 (H3K4). Methylation is co- transcriptional, and H3K4 trimethylation is strongest at the nucleosome nearest the transcription start site, while dimethylation predominates further downstream through the next few nucleosomes. We will explore how Set1/COMPASS is recruited to transcription complexes and how this gradient of methylation is achieved. This aim will also address how levels of COMPASS are controlled. The third specific aim of this project will study the downstream effects of the co-transcriptional histone methylations at H3K4 and H3K36. We will continue our work on the Set3 histone deacetylase complex, which recognizes H3K4me2, and the Rpd3S histone deacetylase complex that recognizes H3K36me2/3. Preliminary data suggests that the Set3 complex strongly affects the kinetics of transcription induction and repression, and several molecular models to explain these effects will be tested. The role of H3K4me3 in recruiting various chromatin modifying complexes will also be explored. The experiments in these three specific aims will significantly increase our understanding of the RNA polymerase II transcription reaction and its interactions with the chromatin template. This fundamental knowledge is essential for understanding how mutations in transcription factors and histone modifying enzymes lead to diseases such as cancer and developmental defects.